Separation and quantification of basic hydroxycinnamic amides and hydroxycinnamic acids by reversed-phase high-performance liquid chromatography

ABSTRACTSimple phenolic acid levels were determined on pooled millstreams of five different classes of Canadian wheat milled to ~75, 80, and 85% extraction. Pooled flours and whole grain were analyzed by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) to establish endogenous levels of insoluble bound, soluble esterified, and free phenolic acids. Only ferulic acid was detected in the insoluble bound category, which accounted for >80% of the total phenolic acids present in every flour. The soluble esterified phenolic acids accounted for up to 17% of the overall total phenolic acid content within a flour. The major constituents were sinapic, ferulic, and vanillic acids, with minor amounts of coumaric, caffeic, and syringic acids. Free phenolic acids accounted for a maximum of 6% of the total phenolic content of any prepared flour. Ferulic acid was the major free phenolic acid, while sinapic acid was not detected in any flour. Significant correlations (r = 0.64–0.97, P < 0.05) were observed between insoluble bound ferulic acid, individual soluble esterified acids, and most free acids with polyphenol oxidase activity, as well as color and ash content for each class.

Mulberries, Morus spp., grow in different growing regions of Turkey with a high diversity providing an opportunity to select superior accessions. The goal of the study was to determine the differences in phytochemical components and horticultural characteristics between white and black mulberry accessions in the Igdir province. Fruits of black and white mulberries collected from Igdir province were assayed for various horticultural characteristics including berry width, berry length, berry weight, and berry peduncle length. Samples were also assayed for vitamin C, organic acids (citric, tartaric, malic, succinic, and fumaric), sugars (glucose and fructose), phenolic acids (catechin, rutin, quercetin, chlorogenic, ferulic, o-coumaric, p-coumaric, caffeic, syringic, vanillic, and gallic acids), and antioxidant capacity [Trolox equivalent antioxidant capacity (TEAC) assay]. Differences (P < 0.05) were noted between accessions in berry length and berry weight. Malic acid was found to be the predominant organic acid regardless of species. Rutin (for black mulberry) and chlorogenic acid (for white mulberry) were the predominant phenolic acids. Black mulberry had higher glucose and fructose content than white mulberry. Analysis showed that black mulberry had higher content of tartaric acid, malic acid, TEAC, fructose, glucose, catechin, rutin, quercetin, o-coumaric acid, and caffeic acid compared with white mulberry (P < 0.01); however, white mulberry had higher content of succinic acid, vitamin C, chlorogenic acid, ferulic acid, p-coumaric acid, syringic acid, and gallic acid (P < 0.01). Our results provide a reference for further studies of mulberry fruits in detection of organic acids, sugars, antioxidant capacity, and phenolic compounds. Also, it is clear that we need standardized testing, extraction, and analysis protocols to compare genotypes grown in different countries.

Hydroxycinnamic acid derivatives (HCAs) are a group of naturally occurring polyphenolic compounds which possess various pharmacological activities. In this work, the interactions of bovine serum albumin (BSA) with six HCA derivatives, including chlorogenic acid (CHA), caffeic acid (CFA), m-coumaric acid (m-CA), p-coumaric acid (p-CA), ferulic acid (FA) and sinapic acid (SA) have been investigated by NMR spectroscopic techniques in combination with fluorescence and molecular modeling methods. Competitive STD NMR experiments using warfarin sodium and L-tryptophan as site-selective probes indicated that HCAs bind to site I in the subdomain IIA of BSA. From the analysis of the STD NMR-derived binding epitopes and molecular docking models, it was deduced that CHA, CFA, m-CA and p-CA show similar binding modes and orientation, in which the phenyl ring is in close contact with protein surface, whereas carboxyl group points out of the protein. However, FA and SA showed slightly different binding modes, due to the steric hindrance of methoxy-substituents on the phenyl ring. Relaxation experiments provided detailed information about the relationship between the affinity and structure of HCAs. The binding affinity was the strongest for CHA and ranked in the order CHA > CFA > m-CA ≥ p-CA > FA > SA, which agreed well with the results from fluorescence experiments. Based on our experimental results, we also conclude that HCAs bind to BSA mainly by hydrophobic interaction and hydrogen bonding. This study therefore provides valuable information for elucidating the mechanisms of BSA-HCAs interaction.

Color enhancing effect of carboxylic acids on anthocyanins

Oat hulls contain relatively high amounts of hydroxycinnamic acids, mainly ferulic (4-hydroxy-3-methoxycinnamic) and p-coumaric acids (4-hydroxy-cinnamic), which are inhibitory to cell wall biodegradability by rumen microorganisms. In this paper, a study of the interactive effects of enriched sources of Aspergillus ferulic acid esterase (A-FAE) and Trichoderma xylanase (T-XYL) at different levels on the quantitative release of ferulic acid and p-coumaric acid from oat hulls was carried out. The results show that relative to A-FAE alone, the combined action of A-FAE and T-XYL was superior in causing the release of ferulic acid [up to 41.0% (± 2.1%)], indicating that T-XYL is important in acting with A-FAE in the degradation of feruloyl-polysaccharides of oat hulls. There was no effect of A-FAE alone, but a significant effect of A-FAE in combination with T-XYL on the release of p-coumaric acid from oat hulls. However, there was no extensive release of p-coumaric acid [(maximum release of 9.0% (± 0.7%)] by A-FAE in the presence of T-XYL, indicating a specificity of A-FAE for feruloyl groups, which only efficiently releases ferulic acid and not p-coumaric acid from oat hulls. This study suggests that A-FAE with T-XYL has an interactive effect to be able to break the ester linkage between ferulic acid and the attached sugar, releasing a significant proportion of the ferulic acid from oat hulls. This action, which causes disruption of crosslinks, has the potential to improve hydrolysis of the remaining polysaccharides by rumen microorganisms, which, in turn, would improve rumen degradability of oat hulls. Key words: Ferulic acid esterase, oat hulls, hydroxycinnamic acids, biodegradation

ABSTRACTArtemisia rutifolia (Asteraceae) had been used in traditional medicines for the treatment of different ailments. In the current study, an effort was made to explore the phenolic composition, antioxidant, and antimicrobial activities of different solvent extracts obtained from A. rutifolia leaves. The reverse-phase high-performance liquid chromatographic (RP-HPLC) analysis revealed the higher extent of polyphenolic compounds (i.e., gallic acid, caffeic acid, chlorogenic acid, syringic acid, sinapic acid, p-coumaric acid, m-coumaric acid, ferulic acid, vanillic acid, myricetin, and quercetin) in methanol extract. Methanol extract consistently showed the highest total phenolic contents (98 ± 2 µg GAE/mg of plant extract), total flavonoid contents (28 ± 0.0 µg QE/mg of plant extract), antimicrobial activity, free radical (DPPH) scavenging (IC50 = 39 µg/mL) activity, and reducing power (18.3 ± 0.2 mg GAE/g of plant extract) followed by those of chloroform and hexane extracts, respectively. The current study concluded that extracts of A. rutifolia are novel natural source of antioxidative and antimicrobial agents for the treatment of oxidative stress-related disorders and microbial infections.

Amycolatopsis sp. ATCC 39116 catabolizes ferulic acid by the non-oxidative deacetylation and β-oxidation pathways to produce vanillin and vanillic acid, respectively. In submerged culture, vanillin productivity decreased more than 8-fold, when ferulic, p-coumaric, and caffeic acids were employed in pre-cultures of the microorganism in order to activate the ferulic acid catabolic pathways, resulting in a carbon redistribution since vanillic acid and guaiacol productivities increased more than 5-fold compared with control. In contrast, in surface culture, the effects of ferulic and sinapic acids in pre-cultures were totally opposite to those of the submerged culture, directing the carbon distribution into vanillin formation. In surface culture, more than 30% of ferulic acid can be used as carbon source for other metabolic processes, such as ATP regeneration. In this way, the intracellular ATP concentration remained constant during the biotransformation process by surface culture (100μg ATP/mg protein), demonstrating a high energetic state, which can maintain active the non-oxidative deacetylation pathway. In contrast, in submerged culture, it decreased 3.15-fold at the end of the biotransformation compared with the initial content, showing a low energetic state, while the NAD+/NADH ratio (23.15) increased 1.81-fold. It seems that in submerged culture, low energetic and high oxidative states are the physiological conditions that can redirect the ferulic catabolism into β-oxidative pathway and/or vanillin oxidation to produce vanillic acid.

Key message4-Coumarate coenzyme A ligase and 4-hydroxybenzoate coenzyme A ligase from the hornwort Anthoceros agrestis expressed in E. coli were characterized on biochemical and molecular levels and showed interesting substrate specificities.Acyl-activating enzymes are associated with the biosynthesis or degradation of various metabolic products such as lipids, amino acids, sugars, and natural compounds. In this work, cDNA sequences encoding 4-coumarate coenzyme A ligase (4CL) and 4-hydroxybenzoate coenzyme A ligase (4HBCL) were amplified from the hornwort Anthoceros agrestis. The coding sequences were expressed in E. coli and purified by Ni-chelate chromatography. The CoA ligases exhibited different substrate specificities. 4CL catalyzed the activation of 4-coumaric acid, 3-coumaric acid, 2-coumaric acid, caffeic acid, isoferulic acid, ferulic acid, and cinnamic acid but lacked activities towards sinapic acid and benzoic acids. In contrast, 4HBCL preferred 4-hydroxybenzoic acid and benzoic acid, but also accepted other benzoic acid derivatives except salicylic acid and 3-aminosalicylic acid. Furthermore, 4HBCL also activated isoferulic acid, cinnamic acid, 2-coumaric acid, 3-coumaric acid, 4-coumaric acid and caffeic acid, but lacked affinity for ferulic acid and sinapic acid. These substrate specificities could be related to the phenolic compounds identified in Anthoceros agrestis.

Mitigation of Pb stress in Scrophularia striata Boiss. by the enhancing phenylethanoid glycoside biosynthesis

Total determination of cinnamic acids (CA), including hydroxycinnamic acid derivatives is generally not accurate since, during hydrolysis, a possible degradation of dihydroxy CA such as caffeic acid could occur. Evaluations of CA (ferulic, p-coumaric, sinapic, cinnamic and caffeic acids) before and after hydrolysis have been undertaken using standards and either with or without addition of ascorbic acid and EDTA. The method was then applied to the determination of free and bound CA in five blond cultivars (Navelina, Washington navel, Pera, Salustiana and Valencia late) of sweet oranges [Citrus sinensis (L.) Osb.]. Four parts of the fruits (peel juice, flavedo, albedo and juice) have been investigated. Results show that CA are mainly bound (86% up to 92%) in the four fruit parts. The mean of total CA contents was found to be higher in peel juice (1.5 g kg(-1)) in comparison with flavedo (0.7 g kg(-1)), albedo (0.1 g kg(-1)) and juice (0.6 g kg(-1)). Free and bound ferulic acid represented 55-70% of CA in juices, followed by p-coumaric acid (20%), sinapic acid (10%) and caffeic acid (9%). Total contents of each CA in the four fruit parts are discussed and show the potential interest in orange peel wastes.

Aromatic compounds are important molecules which are widely applied in many industries and are mainly produced from nonrenewable sources. Renewable sources such as plant biomass are interesting alternatives for the production of aromatic compounds. Ferulic acid and p-coumaric acid, a precursor for vanillin and p-vinyl phenol, respectively, can be released from plant biomass by the fungus Aspergillus niger. The degradation of hydroxycinnamic acids such as caffeic acid, ferulic acid, and p-coumaric acid has been observed in many fungi. In A. niger, multiple metabolic pathways were suggested for the degradation of hydroxycinnamic acids. However, no genes were identified for these hydroxycinnamic acid metabolic pathways. In this study, several pathway genes were identified using whole-genome transcriptomic data of A. niger grown on different hydroxycinnamic acids. The genes are involved in the CoA-dependent β-oxidative pathway in fungi. This pathway is well known for the degradation of fatty acids, but not for hydroxycinnamic acids. However, in plants, it has been shown that hydroxycinnamic acids are degraded through this pathway. We identified genes encoding hydroxycinnamate-CoA synthase (hcsA), multifunctional β-oxidation hydratase/dehydrogenase (foxA), 3-ketoacyl CoA thiolase (katA), and four thioesterases (theA-D) of A. niger, which were highly induced by all three tested hydroxycinnamic acids. Deletion mutants revealed that these genes were indeed involved in the degradation of several hydroxycinnamic acids. In addition, foxA and theB are also involved in the degradation of fatty acids. HcsA, FoxA, and KatA contained a peroxisomal targeting signal and are therefore predicted to be localized in peroxisomes.Key points• Metabolism of hydroxycinnamic acid was investigated in Aspergillus niger• Using transcriptome data, multiple CoA-dependent β-oxidative genes were identified.• Both foxA and theB are involved in hydroxycinnamate but also fatty acid metabolism.

High performance liquid chromatographic (HPLC/UV) method was adapted for simultaneous determination of seven polyphenols, including derivatives of benzoic (gallic and vanillic acids) and cinnamic acids (p-coumaric, ferulic and sinapic acids), flavan-3-ols (catechin) and flavonols (rutin) in worts and beers at the various stages of the brewing process. Based on the semi-quantitative HPLC analysis, total polyphenols chromatographic index (TPCI) was in the ranges of5.18 - 19.4 mg/L and 7.37 - 20.7 mg/L for all worts and beers, respectively. The HPLC analyses showed that relatively high levels of (+)-catechin and gallic acid were in all the worts and the beers, while the values were much lower for ferulic acid, rutin, vanillic acid, sinapic acid and p-coumaric acid. Polyphenols with relatively high concentrations, that were detected in all tested worts and beers, were gallic acid (1.29 - 4.75 mg/L resp. 2.59 - 4.97 mg/L), (+)-catechin (1.66 - 7.95 mg/L resp. 4.70 - 10.0 mg/L) and ferulic acid (0.41 - 4.53 mg/L resp. 1.05 - 2.87 mg/L). On the other side, the sinapic acid(0.72 - 1.59 mg/L resp. 0.72 - 2.5 mg/L), rutin (1.17 - 2.03 mg/L resp. 1.16 - 2.85 mg/L), p-coumaric acid(ND - 4.73 mg/L resp. ND - 1.44 mg/L) and vanillic acid (ND - 1.52 mg/L resp. 0.75 - 1.81 mg/L) were detected in lowest concentrations. In both, worts and beers investigated in this study, the changes in the contents of individual polyphenols were not uniform. In the case of some polyphenols, a decrease in the content was observed after boiling the worts with hops or after the main fermentation until maturation and filtration, but with some polyphenols, the concentrations were constant until the end of the process or even increased.

Phenolic acids are widely distributed in foods and raw materials. They are easily absorbed by humans due to their simplicity. Once they enter the blood plasma, they act as antioxidants. Beer can be a rich source of phenolic acids in the diet. The aim of this study was to determine the concentrations of phenolic acids in two experimental worts and beers as well as in nine market beers (using HPLC-UV). An examination of the total antiradical activities of phenolic acids with in vitro model systems (using ABTS and DPPH free radicals), at the concentrations comparable to those detected in beers, was performed. Only low fractions of the main phenolic acids present in barley malt (ferulic, vanillic and p-coumaric acid) were detected in the experimental worts. Moreover, the concentrations of phenolic acids significantly decreased until the last steps of beer production. The main beer phenolic acids (vanillic and ferulic acid) exerted a lower share of total antiradical activity against both free radicals (calculated as the sum of the individual activities of all acids detected in beer) than the minor phenolic acids (caffeic, chlorogenic, o-coumaric, sinapic or syringic acid). The synergies, between individual phenolic acids in pairs, were also studied with in vitro model solutions using free radicals. The total antiradical activity of the compounds studied in pairs, was at the most as high as the sum of the antiradical activity of the individual phenolic acids, but in most cases it was considerably lower (i.e. no synergy was detected).

High performance liquid chromatographic (HPLC) analysis of culture filtrates of plant growth promoting rhizobacteria (PGPR) and medium of inhibitory zone of interaction of Sclerotium rolfsii with PGPR, viz. Pseudomonas aeruginosa, Pseudomonas fluorescens 4, Pseudomonas fluorescens 4 (new) and Pseudomonas sp. varied from sample to sample. In all the culture filtrates of PGPRs, P. aeruginosa had nine phenolic acids in which ferulic acid (14.52 μg/ml) was maximum followed by other phenolic acids. However, the culture filtrates of P. fluorescens 4 had six phenolic acids with maximum ferulic acid (20.54 μg/ml) followed by indole acetic acid (IAA), caffeic, salicylic, o-coumeric acid and cinnamic acids. However, P. fluorescens 4 culture filtrate had seven phenolic acids in which salicylic acid was maximum (18.03 μg) followed by IAA, caffeic, vanillic, ferulic, o-coumeric and cinnamic acids. Pseudomonas sp. also showed eight phenolic acids where caffeic acid (2.75 μg) was maximum followed by trace amounts of ferulic, salicylic, IAA, vanillic, cinnamic, o-coumeric and tannic acids. The analysis of antibiosis zone of PGPRs showed fairly rich phenolic acids. A total of nine phenolic acids were detected in which caffeic acid was maximum (29.14 μg/g) followed by gallic (17.64 μg/g) and vanillic (3.52 μg/g) acids but others were in traces. In P. aeruginosa, antibiosis zone had seven phenolic acids where IAA was maximum (3.48 μg/g) followed by o-coumeric acid (2.08 μg/g), others were in traces. The medium of antibiosis zone of P. fluorescens 4 and P. fluorescens 4 new had eight phenolic acids in which IAA was maximum with other phenolic acids in traces.

Phenolic compounds in the soils collected from 6 paddy fields and 9 forests were surveyed quantitatively by gas chromatography. The results are as follows: 1) p-Coumaric, ferulic, vanillic, p-hydroxybenzoic, syringic, salicylic, and protocatechuic acids were detected in all soils. Among these acids, p-coumaric acid was present in the largest quantity in most of the soils. Gallic, caffeic, and sinapic acids, resorcinol, and phloroglucinol were not detected. 2) The total amounts of individual phenolic acids in the forest soils (34-632 ppm) were markedly higher than those in the paddy soils (10-26 ppm). p-Coumaric acid content went up to 188 ppm in the forest soils and up to 7 ppm in the paddy soils. The average concentration of total phenolic acids per weight of organic matter in the forest soils was about 2 times as high as that in the paddy soils. 3) In the paddy soils, the concentrations of p-hydroxybenzoic, vanillik, syringic, p-coumarie, and ferulic acids, which have no chelating ability, were considerably related to the pH and the carbon content of the soil, but the concentrations of protocatechuic and salicylic acids, which are chelating agents, were not related to those of the soil. Also in A horizons of forest soils, the concentrations of many phenolic acids were related to carbon content, but not to pH. 4) In a forest soil surveyed, the concentrations of individual phenolics decreased with increasing depth. However, the ratios of the amounts of individual phenolics to the carbon content increased with depth.